Composition from lobster hemocyte extracts for detection of lipopolysaccharides, peptidoglycans and 1,3-beta-D-glucans

ABSTRACT

The present invention relates to the pharmaceutics, biotechnological and chemical, and particularly to a process for preparing a composition for detecting and measuring the concentration of endotoxins or lipopolysaccharides, peptidoglycans and (1,3)-β-D-glucans, using an extract from the hemocytes of the lobster as a starting material, the changes to the composition to increase the sensitivity, and processes for measuring endotoxins, peptidoglycans and (1,3)-β-D-glucans using said composition.

PRIOR RELATED APPLICATIONS

This application is a 371 U.S. National Phase Patent Application whichclaims priority to PCT Patent Application No. PCT/CU2012/000009, filedDec. 27, 2012 and Cuba Patent Application CU/P/2011/0243, filed Dec. 27,2011 and incorporates the above-referenced applications in theirentireties by reference thereto.

DESCRIPTION OF THE INVENTION

The present invention relates to the field of chemical, pharmaceuticaland biotechnology, particularly with the detection of microorganisms andstructures associated to their cell wall such as lipopolysaccharides(LPS) or endotoxins from Gram negative bacteria, peptidoglycans (PG)from Gram negative and positive, and (1,3)-β-D-glucans (BG) from fungiand yeasts. The present invention also has application in the assessmentof microbial contamination in ultrapure water as that used in thesemiconductor industry, and as a diagnostic tool for the clinicallaboratory.

The innate immune system provides very effective response mechanisms forthe detection of microorganisms through recognition of molecularstructures preserved and shared by a great number of microbes. Thesestructures known as pathogen-associated molecular patterns (PAMPs) arenot found in the host and are essential for the survival orpathogenicity of microorganisms. Among the best-characterized PAMPs areLPS, BG and PG, among others [Medzhitov and Janeway, The New EnglandJournal of Medicine, 343(5):338-344, 2000].

It has been long established that endotoxins are the most relevant TAMPor exogenous pyrogen for the pharmaceutical and biotechnologicalindustries because of their remarkable biological potency, ubiquity,resistance to conventional sterilization methods, and high probabilityof contaminating parenteral solutions [Williams K. L. EndotoxinRelevance and Control Overview; in Williams, K. L. (eds). Endotoxins,Pyrogens, LAL Testing and Depyrogenation. Third edition. HealthcareReport, New York, London. 2007, 27-46]. Furthermore, they are the mainresponsible of endotoxic shock associated with sepsis caused by Gramnegative bacteria, and constitute the first alarm signal indicating thepresence of these bacteria to the innate immune system. Access of LPSinto the bloodstream produces profound deleterious effects on humanhealth, such as systemic inflammatory response syndrome (SIRS), multipleorgan failure (MOF), shock, and death [Opal S M, Contrib Nephrol 167,14-24, 2010, Hodgson J C, J Comp Pathol 135, 157-175, 2006].

BACKGROUND OF THE PRIOR ART

The Limulus Amebocyte Lysate test (LAL) has been widely used for thedetection of bacterial endotoxins, especially in the quality control ofparenteral pharmaceutical and biotechnological products (U.S. Pat. No.4,107,077, U.S. Pat. No. 4,279,774, U.S. Pat. No. 4,322,217, U.S. Pat.No. 3,954,663, U.S. Pat. No. 4,038,029, U.S. Pat. No. 4,188,264, U.S.Pat. No. 4,510,241, U.S. Pat. No. 5,310,657).

The LAL reagent is prepared from an extract of amebocytes present in thehemolymph of horseshoe crabs. It consists of a cascade of trypsin-likeserine peptidases, which is activated in presence of endotoxins and(1,3)-β-D-glucans through the Factors C and G, respectively. The reagentis based on the clotting response of horseshoe crabs, which is part ofits innate immune system. A similar system has not been found in otherinvertebrate species so far [Iwanaga S y Lee B L, J Biochem Mol Biol 38,128-150, 2005].

Endotoxin-specific LAL reagents have been obtained by separating FactorG sensitive to (1,3)-β-D-glucans, leaving remaining components of theenzymatic cascade of the LAL (U.S. Pat. No. 5,401,647, U.S. Pat. No.5,605,806, US20030104501) or inhibiting its activation (U.S. Pat. No.5,047,353, U.S. Pat. No. 5,155,032, U.S. Pat. No. 547,984, U.S. Pat. No.5,702,882, U.S. Pat. No. 5,998,389, U.S. Pat. No. 5,179,006).

Horseshoe crabs are marine arthropods known as living fossils becausethey have evolved little in the last 300 million years. There are fourspecies of horseshoe crabs from which a similar reagent can be obtained.

The species Tachypleus tridentatus, Tachypleus gigas and Carcinoscorpiusrotundicauda are exclusively in Asia. The small population of the lasttwo has never sustained production of the reagent. Moreover, Tachypleustridentatus, commonly known as Chinese horseshoe crab or tri-spinehorseshoe crab, had a high population density along the coast of China,especially in the northern South China Sea and in region to the range ofHainan Island. However, recent studies on T. tridentatus in Taiwan,Japan, Hong Kong, Thailand and China, have indicated that populationshave declined drastically almost to extinction. The main causes areoverfishing and pollution of the seas.

Due to the low population density of the Asian species, the LAL reagentmarketed is mostly obtained from Limulus polyphemus, also known as theAmerican horseshoe crab. The Limulus inhabits the US Atlantic coast,from northern Maine to the Yucatan Peninsula and the Gulf of Mexico. Thegreatest population is found in Delaware Bay but there has been adrastic decline in its numbers and spawning activity [Widener J W andBarlow R B, Biological Bulletin (197): 300-302, 1999]. The main causeshave been the habitat loss and use as bait in fishing for oysters andeels [Rudloe A, Journal of Invertebrate Pathology (42):167-176, 1983;Bolton M L, Biologist (49): 193-198, 2002]. Mortality due to bleedingprocess for the preparation of LAL reagent ranged around 15% [Walls E Aand Berkson J, Virginia Journal of Science 51(3):195-198, 2000], but hasdoubled in the past time, increasing the decline of its population.

According to FAO, due to the high price of the LAL reagent, the narrowrange of distribution of horseshoe crabs, and the extremely long time ittakes for them to reach sexual maturity, it is easy to reduce thehorseshoe crab population below the recovery rate. The four species arein danger of extinction and are included on the Red List of theInternational Union for the Conservation of Nature and Natural Resources(IUCN).

Considering the current critical situation of the horseshoe crabpopulation, the United States Atlantic States Maritime FishingCommission (ASMFC) has regulated several aspects concerning fishing andexploitation of the Limulus. Regarding the LAL industry, establishedthat specimens cannot be bled to death and must be returned to thecapture site marked and in healthy condition within less than 72 hours.It also set annual quotas of animals to be used to produce LAL, and thatmortality associated with their handling could not exceed 15% of thisquota.

Globally it has raised the need to study alternatives for the detectionof endotoxins and other pyrogens before the source of raw material, thehemolymph from Limulus, is no longer available. The establishments ofannual quota of horseshoe crabs that can be used by the industry toproduce LAL, while its demand continues on the rise, coupled with therecession of the population, indicate that it is not possible to sustainthe increase in capture and bleeding of horseshoe crabs to produce LALreagent.

U.S. Pat. No. 4,229,541, U.S. Pat. No. 5,082,782, U.S. Pat. No.6,790,659 and US20030186432 describes the development of strategies forthe in vitro culture of amebocytes as an alternative to the hemolymph ofhorseshoe crab as natural source of amebocytes for preparing LAL. Todate there is no commercial reagent obtained in this way or a projectfor its obtaining. On the other hand, through a novel methodology it hasbeen successfully cloned and produced the recombinant of theendotoxin-sensitive Factor C from the Singapore horseshoe crab,Carcinoscorpius rotundicauda. (U.S. Pat. No. 5,712,144, U.S. Pat. No.5,858,706, U.S. Pat. No. 5,985,590), and has been established its usefor the detection and quantitation of LPS or endotoxin in a similarapproach to the natural LAL reagent (U.S. Pat. No. 6,645,724). It havebeen patented reagent formulations that combine the recombinant Factor Cand detergents (US20030054432) or recombinant Factor C or that obtainedby purification from natural source in formulations that exhibitincreased sensitivity and stability (US20040235080). However, due to thelack of other enzymes naturally present in the reagent, theseformulations lack the sensitivity provided by the amplifier effect ofthe enzymatic cascade, which has been partially solved by employing moresensitive fluorescent substrates. Other components of the LAL cascadesuch as the clotting enzyme (US20090208995) and the Factor G(US2010011266) have also been produced recombinant.

The LAL reagent and the recombinant Factor C are able to detect LPS, butdo not detect PG. However, recent studies show the urgent need to alsodetect and control the content of PG in formulations for parenteral use,and other solutions and devices that should be non-pyrogenic. The PG arewell-conserved components of cell wall of Gram positive and negativebacteria, and are the main responsible for the inflammatory response andits deleterious health consequences caused by Gram positive organisms,including septic shock [Silhavy T J et al., Cold Spring Harborperspectives in biology 2, a000414, 2010], As the LPS, the PG are ableto induce the release of proinflammatory cytokines [Verhoef J andMattsson E, Trends in Microbiology 3:136-140, 1995; Teti G, Trends inMicrobiology 7; 100-101, 1999], are pyrogenic, released into theenvironment during growth and death of bacteria, and are resistant toordinary means of sterilization [Moesby L, European Journal ofPharmaceutical Science 35, 442-446, 2008]. Due to the similaritiesbetween LPS and PGs it has been proposed that both be granted the sameimportance [Myhre A E et al., Shock 25 (3):227-35, 2006]. Moreover, thepresence of PG can markedly sensitize the inflammatory response inducedby LPS by synergism [Takada H et al., Journal of Endotoxin Research 8,337-342, 2002; Hadley J S et al., Infection and Immunity, 73; 7613-7619,2005; Wray G M, Shock, 15(2):135-42, 2001; Shikama Y et al., InnateImmunity, 17(1):345, 2009] or by additive effect [Sprong T, Journal ofLeukocyte Biology, 70:283-288, 2001].

Like the LAL cascade for horseshoe crabs, the prophenoloxidaseactivating system (ProPO system) is an essential component of thehumoral innate immune response of invertebrates [Cerenius et al., TrendsImmunol. (29):263-271, 2008]. The ability of proPO system to recognizePAMPs through a biosensor(s) and produce a visible and quantifiableresponse by measuring the enzymatic activity of the prophenoloxidaseactivating enzyme (ppA) or the phenoloxidase, makes it an attractivecandidate for the development of a reagent for the detection ofmicroorganisms and their PAMPs.

The ProPO system comprises a complex array of proteins includingpattern-recognition proteins, the ppA and proPO. It has been describedin several arthropods that the proPO system is activated in the presenceof small amounts of PAMP through a mechanism that has not been fullyelucidated. In general, it is known that in presence of PAMP the pro-ppAbecome active and, through proteolytic attack, converts prophenoloxidase(inactive zymogen) into active phenoloxidase. The phenoloxidase oxidizesmonophenols and/or o-diphenols to aminechromes, initiating the synthesisof melanin.

In U.S. Pat. No. 497,052 it is described the development and use of areagent obtained from the plasma of silkworm larvae (SLP) for thedetection of BG and PG by determining phenoloxidase activity or ppApeptidase activity. It also comprises reagent modifications pursuing thespecific determination of BG or PG. Based on this invention, U.S. Pat.No. 5,585,248 describes the use of synthetic sensitive chromogenicsubstrates for detecting ppA activity. In U.S. Pat. No. 6,274,565 B1 isprotected a modification of the reagent that allows specific detectionof PG by inhibiting the activation pathway mediated by BG. The assay SLPappears to be particularly useful in the detection of bacterialcontamination in platelets (U.S. Pat. No. 7,598,054 B2). Also making useof the proPO system, U.S. Pat. No. 6,987,002 B2 and 2002/0197662 A1describe a reagent obtained from whole hemolymph of insect larva(Tenebrio molitor or Holotrichia diomphalia) for detection andquantification of (1,3)-β-D-glucans.

The proPO system in the spiny lobster P. argus is found in the hemocytes[Perdomo-Morales et al., Fish Shellfish Immunol (23):1187-1195, 2007],and is capable of being activated in the presence of low concentrationsof PG, BG and LPS. There are at least two peptidases related with theactivation of proPO zymogen, one is calcium-dependent and the other not.The proPO system is regulated by a peptidase inhibitor of 5 kDa and netpositive charge. The separation of the inhibitor from the activecomponents of the system substantially increases the sensitivity of theresponse to PG, BG and LPS. The proPO system of the lobster alsoincludes a LPS and BG recognizing protein (LGBP), which is localized inthe plasma fraction of hemolymph. LGBP become activated upon binding toLPS and BG and is capable to increase the phenoloxidase activity in thelysate by increasing ppA activity.

The composition we are presenting here for first time, consist in anaqueous extract of hemocytes from the hemolymph of lobsters, hereafterreferred as Lobster Hemocyte Lysate (LHL). The active component ofinterest in the LHL is the proPO system, and the composition is directedto the detection and quantitation of LPS, PG and BG. The term lobster orlobsters in this document refers to species listed within infraordersAstacidea, Palinura (Achelata) and Thalassinidea, Reptantia (macrura),Order Decapoda, class Crustacea, Phylum Arthropoda.

Although lobsters are also subject to overfishing in some regions, arevery abundant species representing the major fishery resource in manycountries (eg, Cuba, Brazil, Australia, USA, etc.). Production of thepresent composition does not affect the availability of these animalsfor human consumption as food because the hemolymph is a byproduct thatis usually discarded. Lobster is one of the largest crustaceans, andpresents a significant volume of hemolymph readily available.

For the preparation of LHL, the hemolymph is withdrawn using a suitableanticoagulant that prevent plasma coagulation, but preferably that avoidboth plasma clotting as well as the activation and clump of cells. Asanticoagulants can be used an isotonic solution containing methylxanthine derivatives such as theophylline, theobromine or caffeine, orsalts thereof, or reagents capable of modifying sulfhydryl groups (—SH)as cysteine, iodoacetamide and N-ethylmaleimide. Also suitable aresolutions containing chelating agents, having a pH between 4.5-8provided by a suitable buffer, for example, the modified Alseveranticoagulant (27 mM sodium citrate, 336 mM NaCl, 115 mM glucose, 9 mMEDTA, pH 7) or citrate-EDTA anticoagulant (0.4 M NaCl, 0.1 M glucose, 30mM trisodium citrate, 26 mM citric acid and 10 mM EDTA, pH 4.6).Modified Alsever anticoagulant is preferred, using a proportionhemolymph:anticoagulant 1:1 (v/v).

The hemocytes are separated from plasma by centrifugation at 700 g for10 min at 4° C. The plasma (supernatant) is discarded, and thesedimented hemocytes are washed to remove residual plasma components.For this purpose, the hemocytes are resuspended with anticoagulant to avolume no greater than that corresponding to the initial mixture ofhemolymph:anticoagulant, followed by another identical centrifugationcycle. The hemocytes should be washed at least twice. The washedhemocyte pellet is suspended in lysis buffer, preferably with a volumebetween 1 and 10 ml. The lysis buffer may contain NaCl at aconcentration between 0.001 and 600 mM, agents capable of stabilizingenzymes (stabilizers), and a pH between 5 and 8.5 provided by a suitablebuffer that does not sequester divalent cations. The lysis buffer 50 mMTris-HCl, pH 7.5, 450 mM NaCl is preferred.

The hemocytes are lysed using a suitable disruption method among thosecommonly used in biochemistry for preparation of cell extracts, whichcan be mechanical, chemical or enzymatic. Considering thecharacteristics of these cells, the most suitable rupture methods areosmotic shock, freeze/thawing, homogenization by stirring (vortex) ormanual (bounce and Potter-Elvehjemem), and ultrasonication. Thehemocytes homogenate is centrifuged at 13 000 rpm at 4° C. for 30 min toobtain the clarified supernatant or LHL.

The present invention includes a modification for increasing thesensitivity of the LHL response to LPS, PG and BC. This modification isbased on eliminating or inactivating peptidase inhibitors present in theLHL. The inhibitors can be removed by separation techniques based onmolecular size and shape, charge or affinity. Preferably it will beemployed procedures based on size exclusion as gel filtrationchromatography, ultrafiltration and diafiltration. For gel filtrationchromatography, resins with an exclusion limit between 10 and 60 kDashould be used, preferably 30 kDa. The chromatographic fractionation isperformed at a flow rate between 3 and 60 cm/h, preferably at 9 cm/h,and the sample volume applied should be between 1 and 5% of the totalcolumn volume, preferably 3%. Under these conditions, the remainingactive proteins of the proPO system elutes at the void volume of thecolumn. When using ultrafiltration and diafiltration, membranes withcutoff between 5 and 60 kDa are used, preferably between 10 and 40 kDa.The LHL without inhibitor is hereinafter referred to as modified LHL(LHL-M).

Both reagents, LHL and LHL-M are used for the detection of PG, BC andLPS. This detection is based on the activation of the proPO system inlobster (FIG. 1). Therefore, the detection of PG, BC and LPS isperformed by determining the enzyme activity of the active phenoloxidase(FO) or the peptidase activity of ppAs (FIG. 1).

The phenoloxidase activity is determined using monophenolic substrates(eg tyramine, L-tyrosine, 4-hydroxyphenylacetic acid,4-hydroxyphenylpropionic acid) or diphenolic substrates (egdihydroxy-L-phenylalanine (L-DOPA), dopamine, 3,4-dihydroxyphenylaceticacid, 3,4-dihydroxyphenylpropionic acid, catechol and methyl catechol.It is determined preferably with 1.2 mM dopamine. The formation ofcolored products allows the phenoloxidase activity can be determinedvisually or spectrophotometrically.

The sensitivity of the method to determine the phenoloxidase activitycan be increased by the combination of monophenolic or o-diphenolicsubstrates with 3-methyl-2-benzothiazoline hydrazine (MBTH) orBesthorn's hydrazone. The MBTH is a powerful nucleophilic agent thatforms a stable coloured adduct with o-quinones generated byphenoloxidases (MBTH-quinone), with an extinction coefficient or molarabsorptivity much larger than the corresponding aminechrome [Espin J Cet al., Eur J Biochem, 267:1270-1279, 2000; Garcia-Molina F et al., J.Agric. Food Chem 55:9739-9749, 2007]. The MBTH is used at a finalconcentration between 0.3 to 15 mM, preferably 2 to 7 mM.

The peptidase activity of ppA induced by BG, LPS and PG is determinedusing chromogenic or fluorogenic substrates for trypsin-like serineproteases. These substrates are of general formula R-Y or R-Arg-Lys-Y,where Y is a chromophore like p-nitroaniline (p-NA) or a fluorophoresuch as 7-amido-4-methyl coumarin, rhodamine, or7-amido-4-trifluoro-methylcoumarin. R represents an L or D amino acidprotected by its N-terminus by a protecting group, or a peptidecomprising L or D amino acids, or their combination, protected at itsN-terminus by a protecting group.

Assays for detection of PG, LPS and BG using the LHL or LHL-M reagentcan be performed by kinetic, pseudo-kinetic or endpoint methods. Thereaction can be detected by using conventional spectrophotometers andfluorimeters. However, due to higher sample throughput and saving ofreagents, is preferred the using of microplate reader capable of reading96 well microplates, equipped to read wavelengths in the visible,fluorescence, or those capable of performing both functions.

Kinetic assays are defined as the product detection in time immediatelyafter the addition of all components of the reaction mixture includingthe substrate. Pseudo-kinetic assays are defined as those where theresponse is determined after substrate addition to a previouslyincubated reaction mixture. The endpoint assays are defined as thesingle reading of the response after a fixed time incubation of thereaction mixture with the substrate. The reaction mixture should have apH value between 6 and 9, preferably 7.5, and may or may not containdivalent cations. It should contain calcium, magnesium or manganese in aminimum concentration of 5 mM, preferably 50 mM. The test temperatureshould be between room temperature and 45° C., preferably 37° C.

The present invention also comprises the use of LGBP to increase thesensitivity of both LHL and LHL-M to BG and LPS. The LGBP may beincluded in the formulation of the reagent, or can be added later to thereaction mixture. The LGBP may be in a concentration range in the assayfor the detection of LPS and BG between 3 to 200 μg/ml, preferably from50 to 125 μg/ml.

The LGBP is obtained from plasma, which is also a plentiful byproductfrom the preparation of LHL. After an isoelectric precipitation step[Vargas-Albores F et al., Comp Biochem Physiol B Biochem Mol Biol,116(4):453-458, 1997], the LGBP can be purified by affinitychromatography using hydrophilic matrices bound to (1,3)-β-D-glucans[Vargas-Albores F et al., Comp Biochem Physiol B Biochem Mol Biol, 116(4): 453-458, 1997], immunoaffinity [Duvic B and Söderhäll K, J Biol.Chem., 265(16): 9327-9332, 1990] or heparin [Jimenez-Vegas F et at, FishShellfish Immunol 13(3):171-181, 2002]. Preferably, heparin is used asligand, allowing the obtaining of large amounts of pure LGBP in a singlechromatographic step.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic representation of the prophenoloxidase activatingsystem (proPO system) in the spiny lobster, and principle for thedetection of lipopolysaccharides (LPS), peptidoglycans (PG) and(1,3)-β-D-glucans (BG) using the Lobster Hemocyte Lysate (LHL). LGBP:Lipopolysaccharide and (1,3)-β-D-glucans binding protein.

FIG. 2. Influence of concentrations of o-diphenolic substrates dopamineand L-DOPA on phenolixadase activity in the Lobster Hemocyte Lysate.

FIG. 3. LHL response to lipopolysaccharides (LPS), (1,3)-β-D-glucans(BG) and peptidoglycans (PG). Panels A-C: phenoloxidase activity inarbitrary units (Δ OD 490 nm/min). Panels D-F presents lineal relationbetween phenoloxidase activity and microbial elicitor concentration. LPS(0.185-1850 ng/ml), BG (1.8-18000 ng/ml) and PG (0.19-19000 ng/ml).Onset time means the reaction time required to reach a specific opticaldensity at 490 nm.

FIG. 4. Influence of the MBTH in the sensitivity of phenoloxidaseresponse. Left panel: Optical density at 490 nm after 1 h at 37° C.(endpoint assay). Right panel: Reaction rate in absence (Control) andpresence of different concentrations of MBTH.

FIG. 5. Determination of proPO system response to LPS using chromogenicsubstrate for serine peptidase S-2222(Bz-Ile-Glu(γ-OR)-Gly-Arg-pNA-HCl).

FIG. 6. Separation of protease inhibitor from the active components ofproPO system by gel filtration chromatography in a Sephadex G-50 column.

FIG. 7. Phenoloxidase response of LHL lacking protease inhibitor usingdopamine as substrate. Sensitivity of 0.01 ng/ml LPS.

FIG. 8. Purification of LGBP by affinity chromatography inheparin-Sepharose CL-6B (Panel A). SDS-PAGE Electrophoresis (Panel B):Molecular weight marker (lane 1), whole plasma (lane 2), purified LGBPunder reducing (lane 3) and non-reducing (lane 4) conditions. Proteinswere stained with Coomassie Brilliant Blue R-250.

EXAMPLES Example 1

Preparation of Lobster Hemocyte Lysate (LHL)

Ten milliliters of hemolymph were obtained from the fourth walking legcoxa using 20 ml sterile and pyrogen-free disposable syringes containing10 ml of cold anticoagulant solution. The anticoagulant used was Alsevermodified solution composed by 27 mM sodium citrate, 336 mM sodiumchloride, 115 mM glucose, 9 mM EDTA, pH 7 (1000 mOsmol). The mixture ofhemolymph:anticoagulant was poured into 50 ml sterile and pyrogen-freepolypropylene centrifuge tubes and centrifuged at 700 g and 4° C. for 10min. The supernatant containing plasma was discarded. Thereafter thepelleted hemocytes were washed to remove residual plasma components. Toaccomplish this, the hemocyte pellet was suspended with 20 ml of coldanticoagulant, and then the volume was raised up with anticoagulant tothe original anticoagulant:hemolymph volume (50 ml), and centrifugedagain as described above. The washing step was repeated once more. Thewashed hemocytes pellet was resuspended in 3 ml of lysis buffer composedby 50 mM Tris-HCl, 450 mM NaCl, pH 7.5, and then transferred to 13 mm×10cm borosilicate tubes. The hemocyte suspension was lysed by sonicationusing three cycles of 20 Watts for 10 seconds in ice bath. The hemocytehomogenate was centrifuged at 13 000 rpm for 30 min. at 4° C. to obtainthe clarified LHL.

Example 2

Comparing the Effect of Dopamine and L-DOPA Concentrations in theSensitivity of Phenoloxidase Response in the LHL

The lysate (420 μl at 0.1 mg/ml) was incubated with 4.2 ml of 1 mg/mlbovine trypsin for 30 min at 37° C. Hundred microliters of this mixturewere added to the wells of a microplate containing 150 μl of variousconcentrations of L-DOPA (0.3 to 8 mM) or dopamine (0.15 to 9 mM) in 50mM Tris-HCl, pH 7.5. The phenoloxidase activity was determinedkinetically at 490 nm for 20 min at 37° C. in a microplate reader.

Example 3

Determination of PG, BG and LPS Using the LHL Through Detecting thePhenoloxidase Activity by Kinetic Method

The LHL (12 mg/mg was diluted to 0.5 mg/ml with 50 mM Tris-HCl, pH 7.5.The assay was performed in 96-well flat-bottom microplates that weresterile and pyrogen free certified. The reaction mixture consisted of150 μl of 50 mM Tris-HCl, pH 7.5, 50 mM CaC₂, 20 μl of LHL, and 50 μl ofactivator (LPS, PG or BG). Control was endotoxin free water. Finally, 50μl of 3.75 mM dopamine was added as substrate. Dopaminechrome formationwas assessed kinetically at 490 nm during 1 h by reading every 15 sec at37° C. in a microplate reader. The reaction velocity (ΔOD 490/min) wasdetermined from the linear portion of the plot between OD 490 nm vs.time. Alternatively, the onset time was determined as the time requiredfor the reaction mixture to reach an absorbance value of 0.1. The plotsof the logarithm of the onset time vs. the logarithm of PAMPconcentration were lineal and useful for quantitative purposes.

Example 4

Effect of MBTH in Increasing the Phenoloxidase Response in the LHLTitration

The LHL at 0.8 mg/mL was diluted to 0.1 mg/mL with 50 mM Tris-HCl, pH7.5. The assay was performed in flat-bottom-well microplates. Thereaction mixture consisted of 40 μl of 50 mM Tris-HCl, pH 7.5, 160 μl 1mg/ml trypsin dissolved in 50 mM Tris-HCl, pH 7.5, 10 μl of LHL and 40μl of MBTH dissolved in distilled water. This mixture was incubated for20 minutes at 37° C. and finally 50 μl of 3.75 mM dopamine were added toeach well. The reaction was kinetically read at 490 nm for 1 h every 15sec at 37° C. in a microplate reader.

Example 5

Determination of LHL Response Against LPS Using Chromogenic Substratesfor Serine Peptidases

Fifty microliters of LHL (1 mg/ml) obtained as described in Example 1,but without sodium chloride in the lysis buffer, were combined with 150μl of 50 mM Tris-HCl, pH 7.5 buffers, and 50 μl of LPS. Thereafter, 50μl of 0.6 mM chromogenic substrate S-2222 (Bz-Ile-Glu(γ-OR)-Gly-Arg-pNA-HCl) were added to each well. The p-nitroanilinereleased was kinetically detected at 405 nm for 1 h at 37° C.

Example 6

Protease Inhibitor Separation by Gel Filtration Chromatography

The protease inhibitor is separated from the active components of theproPO activating system in the LHL by using gel filtrationchromatography on Sephadex G-50 superfine packed in a column XK 16/70(Vt=131.4 ml) that was previously equilibrated with 50 mM Tris-HCl, 0.2M NaCl, pH 7.5. Four milliliters of 8 mg/ml LHL (3% Vt) werefractionated at a flow rate of 0.3 ml/min. The chromatographicfractionations were monitored at 280 nm. The fraction corresponding tothe void volume of the column was collected and designated as F1 ormodified LHL (LHL-M).

Example 7

Protease Inhibitor Separation by Spin Column

The LHL was fractionated on a spin column packed with Sephadex G-50 (1.5ml). The column was equilibrated with 50 mM Tris-HCl, 450 mM NaCl, pH7.5 buffers by washing the column 4 times at 1000 rpm for 1 min. Onehundred and fifty microliters of LHL, prepared as in Example 1, wereapplied to the spin column and centrifuged at 1000 rpm for 1 min. Theeluate was collected, and both the LHL (also named FO fraction) and theeluate or fractionated LHL (also named F1 or LHL-M) were analyzed forprotein concentration, inhibitory activity against trypsin, andphenoloxidase activity.

Protein Concentration F0 150 μl 9.7 mg/ml F1 150 μl   3 mg/ml InhibitoryActivity (IA) IA/ml IA/mg F0 2378.81 ± 258.938  225.55 ± 36.586 F1 19.66± 0.277  6.598 ± 0.093 Phenoloxidase Activity (PO) PO/ml PO/mg F0 0.265± 0.0240  1.366 ± 0.01240 F1 0.089 ± 0.0090  1.483 ± 0.01650

Example 8

Determination of Phenoloxidase Response Against LPS in LHL LackingTrypsin-Inhibitory Activity or LHL-M

The lysate without inhibitor (Modified LHL; LHL-M) was obtainedaccording to Example 5. The reaction mixture with 50 of LPS, 150 μl ofTris-HCl, 50 mM CaCl₂, pH 7.5, and 20 μl of 0.8 mg/ml lysate (LHL-M)were incubated at 37° C. for 30 min. Finally, 50 μl of 3.75 mM dopaminewas added and the reaction progress was kinetically read at 490 nm for 1hour at 37° C. The onset time was calculated as the time required toreach an optical density of 0.3, then the log of onset time vs. log ofLPS concentrations was plotted.

Example 9

Purification of Lipopolysaccharide and β-1,3 Glucan Binding Protein(LGBP) from Plasma

The plasma obtained during the LHL preparation was centrifuged forclarification at 5000 rpm for 20 min at 4° C. Hundred milliliters ofclarified plasma were dialyzed vs. 5 L of distilled water at 4° C. for48 hours using a dialysis membrane with a cutoff of 8000 Da. Fourchanges of dialyzing water every 12 hours were made during the period.The dialysate was centrifuged at 5000 rpm for 20 min at 4° C. and thesupernatant was discarded. The protein pellet was suspended in 50 mMTris-HCl, 0.2 M NaCl, pH 7.5 (Buffer A), and centrifuged again to removethe insoluble denatured proteins. Thirty-five milligrams of sample in 5ml were applied to a column packed with Heparin-Sepharose CL-6Bpreviously equilibrated in buffer A. After the elution of unboundproteins, the column was washed with 2.5 column volumes of a mixture ofBuffer A:Buffer B (60:40 v/v). Buffer B was composed by 50 mM Tris-HCl,1 M NaCl, pH 7.5. Finally, LGBP was eluted with 5 column volumes withBuffer A:Buffer B (30:70 v/v). The chromatographic process was performedat a flow rate of 1 ml/min and the protein fractions were detected at280 nm.

The invention claimed is:
 1. A process for detecting one or morepathogen associated molecular patterns (PAMPs) selected from the groupconsisting of lipopolysaccharides, peptidoglycans and 1,3-β-D-glucans ina sample, said process comprising the steps of: providing a lysate oflobster hemocytes containing the prophenoloxidase activating (ProPO)system; wherein said ProPO system in said lysate can be is activated byeach one of said PAMPs upon contact; modifying said lysate by removingor inactivating peptidase inhibitors present in said lysate, to therebyprovide a modified lysate of lobster hemocytes containing the ProPOsystem; contacting a sample with said modified lysate of lobsterhemocytes containing the ProPO system, to form a reaction mixture; anddetecting one or more of said PAMPs in said sample by measuring in saidreaction mixture the peptidase activity of the prophenoloxidaseactivating enzyme or the activity of the phenoloxidase enzyme of saidProPO system; wherein said lysate of lobster hemocytes is obtained froma lobster in the infraorders Astacidea, Palinura (Achelata) orThalassinidea; and wherein said step of modifying said lysate comprisesthe removal or inactivation of a trypsin-like peptidase inhibitor havinga molecular weight of about 5 kDa.
 2. The process according to claim 1,wherein the peptidase activity of the prophenoloxidase activating enzymeis determined with a chromogenic or fluorogenic substrate fortrypsin-like serine proteases.
 3. The process according to claim 1,wherein the peptidase activity is measured spectrophotometrically bykinetic, pseudo-kinetic or endpoint methods.
 4. The process according toclaim 1, wherein said step of contacting further comprises the step of:adding to the reaction mixture an amount of between 3 and 200 μg/ml oflipopolysaccharide and 1,3-β-D-glucan binding protein (LGBP), isolatedfrom the plasma of a lobster, so as to substantially increase thesensitivity of detection of lipopolysaccharides and 1,3-β-D-glucans. 5.The process according to claim 1, wherein said step of measuring in saidreaction mixture the peptidase activity of the prophenoloxidaseactivating enzyme or the activity of the phenoloxidase enzyme of saidProPO system comprises kinetic, pseudo-kinetic or endpoint methods. 6.The process according to claim 1, wherein the activity of thephenoloxidase enzyme is measured using monophenolic or o-diphenolicsubstrates.
 7. The process according to claim 6, wherein themonophenolic or o-diphenolic substrates further comprises3-methyl-2-benzothiazolinone hydrazone (MBTH) in concentrations between0.3 and 10 mM in the reaction mixture to increase the sensitivity of thephenoloxidase response.
 8. The process according to claim 1, whereinsaid step of modifying said lysate comprises the removal of saidpeptidase inhibitor based on separation by molecular size and shape,affinity, immunoaffinity, or molecular charge.
 9. The process accordingto claim 1, wherein said lysate of lobster hemocytes is a lysate ofspiny lobster hemocytes.
 10. The process according to claim 1, whereinsaid lysate of lobster hemocytes is a lysate of Panulirus argushemocytes.